Over the last decade, much effort has been dedicated to the development of efficient gene or nucleic acid delivery technologies for introduction and proper expression of genes or nucleic acids in target cells. Therapeutic genes or nucleic acids can be used to restore malfunctioning genes to treat genetic disorders, to induce an immune response to treat cancer and infectious diseases or to suppress an immune response e.g. for inducing/restoring immune tolerance to prevent transplant rejection or to treat autoimmune diseases and allergies. The therapeutic genes or nucleic acids can be administered as naked molecules or as nucleic acids packaged in lipid and/or proteinaceous compounds.
Since viruses evolved to deliver and express their genetic information into their host target cells, viral vectors have been explored as gene delivery vehicles and were found to be by far the most effective means of delivering genetic information into a living cell. A number of viral vector gene delivery systems have been developed and tested in preclinical and clinical trials. These trials revealed that the currently used vectors, which are derived from adenoviruses, poxviruses, herpesviruses, alphaviruses, retroviruses, parvoviruses and polyomaviruses, are generally safe to use and efficient in delivering therapeutic genes to target cells.
A major disadvantage of the currently used viral gene delivery vectors is the fact that they cannot be produced in sufficient amounts to treat significant numbers of patients. The majority of viral vectors is produced by transfecting producer cells with plasmid DNA encoding the vector and the vector components. This generally yields 1 to 10 million vector particles per milliliter cell culture volume. In clinical trials, generally 1×1010 to 1×1012 vector particles have to be administered to a patient in order to accomplish beneficial clinical effects. This means that in order to treat 1000 patients, more than 1 million liters of cell culture are required to yield sufficient amounts of vector particles.
In addition, preclinical and clinical trials revealed that most of the tested viral gene delivery vectors such as adenoviral, poxviral, herpesviral, alphaviral and retroviral vectors induce a strong immune response in patients, directed to viral vector components and the therapeutic gene products. As a consequence these vectors can only be administered a single time to a patient, whereas the expression levels of the introduced therapeutic gene rapidly decline. Viral vectors derived from adeno-associated virus (AAV) do not induce immune responses in animals and are immunologically inert. However, the majority of the human population encountered wildtype AAV together with its helper virus, e.g. adenovirus and as a result developed a strong CTL memory against the AAV capsid proteins. As a consequence AAV-transduced cells are rapidly removed and the expression levels of the therapeutic gene or nucleic acid introduced by an AAV viral vector rapidly decline.
The yields of recombinant proteins produced in mammalian cells compared to those produced in prokaryote cells are in general low, despite the use of strong promoters and/or multicopy transgene insertions or other ways to enhance the transcription. Viral replication competent vectors or replicons have been used for a long time as expression systems for the production of recombinant proteins in mammalian cells. The target gene in such vectors can be expressed under transcriptional control of viral promoters whereby the desired mRNAs may accumulate to extremely high levels in the cytoplasm early after transfection, yielding large amounts of target protein. So far the successes with replicon-based expression systems have been limited. Replicon systems based on RNA viruses in general produce recombinant proteins for only a short period of time, whereas those derived from DNA viruses in general do not replicate well in the commercially used cell lines.
To our knowledge, there is only one viral gene delivery vector that is immunologically inert in humans and that can be produced in sufficient amounts to treat a significant number of patients. Moreover, this viral gene delivery vector can be employed as a replicon system for the production of recombinant proteins in mammalian cell lines. This viral vector system is derived from simian virus 40 (SV40), a simian polyomavirus.
Polyomaviruses are comprised of a family of non-enveloped DNA viruses with icosahedral capsids. They are isolated from a variety of animal species including humans, monkeys, rodents and birds. Five human polyomaviruses have been described, termed BK, JC, WU, KI and Merkel Cell polyomavirus. Many monkey polyomaviruses have been described of which SV40 is the most well-known. SV40 replicates poorly in human cells and infections in humans are rare. Occasional SV40 infections occurred through transmission of the virus from monkeys to people living in close contact with these animals or through vaccination with batches of inactivated poliovirus particles contaminated with SV40.
SV40 has a 5.25 kilo base pairs long circular double stranded DNA genome. The SV40 genome consists of two regulatory regions, the promoter/origin region and the polyadenylation region. The promoter/origin region is 500 base pairs long and comprises two oppositely-directed promoters, the early and late promoter (SVEP and SVLP respectively), the origin of replication and the packaging signal. The polyadenylation region is 100 base pairs long and contains the polyadenylation signals of both the early and the late transcripts. SVEP drives expression of the early primary transcript that is spliced by host-encoded splicing factors into 2 different mRNAs encoding small and large tumor (T) antigens.
The large T antigen is the replicase-associated protein required for DNA replication and for activation of the SVLP. Although the precise role of the small T antigen in virus replication has remained unclear, small T antigen is required for the transformation of several mammalian cell types, in conjunction with large T antigen. The primary effects of small T antigen occur through its interaction with serine-threonine protein phosphatase 2A. The phosphatase 2A-binding domain of small T antigen is located at the unique carboxy-terminal end of the small T antigen.
It is well documented in the prior art that both large T antigen and small T antigen are required for efficient polyomavirus replication (Fahrbach K. M. et al., Virology 370 (2): 255-263, 2008).
SVLP drives expression of the late primary transcript that is spliced by host-encoded splicing factors into different mRNAs encoding the viral capsid proteins VP1, 2 and 3. The T antigens are the major and the capsid proteins the minor immunogenic components of polyoma viruses, eliciting cellular and humoral immune responses against SV40-infected cells.
The SV40 T antigens cooperatively immortalize primary mammalian cells, transform established mammalian cell lines and induce tumours in immuno-compromized young-borne rodents. A number of reports suggest that SV40 infections are associated with human malignancies, caused by the oncogenic activity of the chronically expressed T antigens (Butel J. S. and Lednicky J. A. Journal of the National Cancer Institute 91: 119-134, 1999).
Since expression of the viral capsid proteins is dependent on the presence of the large T antigen, T antigen-specific sequences have been deleted in polyoma viral vectors, not only for rendering the vectors replication-incompetent, but also to completely eliminate their immunogenicity in humans.
T antigen-deleted polyoma viral vectors derived from SV40 have been made and tested, in which the therapeutic genes or nucleic acids are expressed in trans in target cells under transcriptional control of the viral SVEP. Said vectors are known for a long time as potential vectors for gene transfer into a plurality of human tissues and cell types, for example, bone marrow (Rund D. et al, Human Gene Therapy 9: 649-657, 1998), the liver (Strayer D. S. and Zern M. A., Seminars in Liver Disease 19: 71-81, 1999) and dendritic cells (Vera M. et al., Molecular Therapy 12: 950-959, 2005).
Polyomaviral vectors, such as SV40, are known to infect non-dividing as well as actively dividing cells. Since the vectors lack the region encoding the T antigens and as a consequence do not express the viral capsid proteins, they are non-immunogenic (Strayer D. S. and Zern M. A., Seminars in Liver Disease 19: 71-81, 1999) allowing repeated administration to the same individual. Moreover, since the inserted therapeutic gene constructs are expressed under transcriptional control of SVEP, a weak but constitutive promoter, said vectors induce long-term expression of the therapeutic proteins in vivo. Thus, it is known that polyomaviral vectors, such as SV40-derived vectors are promising candidates for therapeutic gene or nucleic acid transfer that can be used for the above mentioned applications.
Because of their replication potential, polyomavirus-based replicons are also of great interest to enhance the production of recombinant proteins such as antibodies, growth factors and hormones in mammalian cells.
T antigen-deleted SV40 particles have been produced in simian cells that are permissive for lytic growth of SV40 and that supply the T antigens in trans. SV40 vector packaging cell lines that are currently used are COS cell lines in particular COS-1 and COS-7 (Gluzman Y., Cell 23: 175-182, 1981). COS cell lines were generated by transformation of monkey CV1 cells with SV40 DNA. Another cell line that expresses the SV40 T antigen in trans is CMT4. The CV1-derived CMT cell lines were generated using SV40 DNA in which the T antigens were expressed under transcriptional control of the mouse metallothionein promoter (Gerard R. D. and Gluzman Y., Molecular and Cellular Biology 5: 3231-3240, 1985).
There is an important disadvantage however to the use of such cell lines. Passaging of T antigen-deleted SV40 vectors in the constructed packaging cell lines (COS or CMT) in many cases results in the appearance of wildtype replication-competent SV40 particles (Gluzman Y., Cell 23: 175-182, 1981; Oppenheim A. and Peleg A., Gene 77: 79-86, 1989; Vera M. et al., Molecular Therapy 10: 780-791, 2004).
This most likely occurs by nucleotide sequence homology-dependent recombination between the chromosomally inserted SV40-specific sequences and nuclear SV40 vector-specific sequences. The emergence of the replication competent wildtype virus particles and the presence of the T antigen oncoproteins in such conventional packaging cell lines have made the use of SV40 vectors for medical purposes impractical.
The human embryo kidney 293 (HEK293) cell line is semi-permissive to SV40 infection, which means that only a small percentage of infected cells support virus replication. The majority of cells are persistently infected and show very low levels of virus replication.
A derivative of the HEK293 cell line is the HEK293T cell line, expressing the SV40 early region under transcriptional control of the Rous sarcoma virus long terminal repeat promoter. It has been described that HEK293T cells express very low amounts of large T antigen and large amounts of small T antigen, due to a splicing bias in favour of the SV40 small T antigen mRNA. Vera et al. found that HEK293T poorly supports SV40 viral vector production (Vera M., et al., Molecular Therapy 10: 780-791, 2004).
Since the T antigen oncoproteins are present in HEK293T cells and there is a risk that replication competent SV40 viruses emerge, the use of this cell line for the production of SV40 vectors for medical purposes is undesired and impractical.
The HEK293TT cell line has been developed as a derivative of HEK293T, generated by stable transfection with a gene construct encoding the SV40 large T antigen. HEK293TT cells are used for the production of recombinant human papilloma virus (HPV) pseudo-vector particles. The recombinant HPV pseudo-vector particles are produced in HEK293TT by transfecting the cells with a plasmid that harbours the SV40 origin of replication and the HPV capsid genes and one that harbours the SV40 origin of replication and a HPV pseudo-genome (Buck C. B. et al., Methods in Molecular Medicine 119: 445-462, 2005).
Since HEK293TT as a derivative of HEK293T accumulates the small and large T antigen oncoproteins and poorly supports SV40 replication, the use of this cell line to produce recombinant SV40 vectors for medical purposes is also undesired and impractical.
WO 03/025189 describes packaging complementation cell lines that allow for the production of SV40 vector particles that are allegedly safe for medical use. However, the packaging cell lines described herein still accumulate significant amounts of the small and large T antigen oncoproteins.
Vera M. et al., Molecular Therapy 10: 780-791, 2004 showed that the production capacity of recombinant SV40 vector particles of interest in certain cell lines such as CMT4 and HEK293T can be very low and state that the cell lines described in WO 03/025189, such as COT-2 are also not effective as producer cell lines for the recombinant SV40 virus particles, possibly due to the splicing bias in favour of the small T antigen mRNA in these cell types.
WO 08/000779 describes a method to overcome the problem with the production of high titre stocks of suitable SV40 viral vectors using viral suppressors of RNA interference (RNAi), such as the vaccinia virus E3L and influenza A virus NS1 proteins. The packaging cell lines described in WO 08/000779 do not provide a solution to the disadvantages of the packaging cell lines of the prior art described herein above.
Chinese hamster ovary (CHO) cells have been provided with the mouse polyomavirus early region, resulting in CHOP cell lines (Heffernan and Dennis, Nucleic Acids Research 19: 85-92, 1991). A number of CHOP cell lines supported replication of plasmid CDM8 (invitrogen), a mammalian expression vector carrying the mouse polyomavirus origin of replication. The level of replication in the CHOP cell lines was not sufficient to make this system attractive for commercial application, possibly due to a splicing bias in favour of the small T antigen or middle T antigen mRNA in CHO cells.
There remains a desire in the art for efficient production systems for recombinant polyomavirus particles that are safe to use and yield high titers of viral vector particles. It is therefore an object of the present invention to provide methods for the safe and efficient production of polyomavirus particles and compositions obtainable therewith. It is appreciated that the methods of the present invention can also be used for the production of large amounts of recombinant proteins in mammalian cells.